Determination by gel filtration

Determination of molecular mass of pectic enzymes The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with Tris-HCl 50 mM, pH 7,5 buffer, plus 100 mM KCl. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were tyroglobulin (660 kDa) apoferritin (440 kDa) P-amylase (200 kDa) alcohol dehydrogenase (150 kDa) bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were myosin (205 kDa) p-galactosidase (116 kDa) phosphorylase b (97 kDa) bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). 

The overall distribution of lanthanides in bone may be influenced by the reactions between trivalent cations and bone surfaces. Bone surfaces accumulate many poorly utilized or excreted cations present in the circulation. The mechanisms of accumulation in bone may include reactions with bone mineral such as adsorption, ion exchange, and ionic bond formation (Neuman and Neuman, 1958) as well as the formation of complexes with proteins or other organic bone constituents (Taylor, 1972). The uptake of lanthanides and actinides by bone mineral appears to be independent of the ionic radius. Taylor et al. (1971) have shown that the in vitro uptakes on powdered bone ash of 241Am(III) (ionic radius 0.98 A) and of 239Pu(IV) (ionic radius 0.90 A) were 0.97 0.016 and 0.98 0.007, respectively. In vitro experiments by Foreman (1962) suggested that Pu(IV) accumulated on powdered bone or bone ash by adsorption, a relatively nonspecific reaction. On the other hand, reactions with organic bone constituents appear to depend on ionic radius. The complexes of the smaller Pu(IV) ion and any of the organic bone constituents tested thus far were more stable (as determined by gel filtration) than the complexes with Am(III) or Cm(III) (Taylor, 1972). 

The cell-bound amylopullulanase was solubilized with detergent and lipase. It was then purified to homogeneity by treatment with streptomycin sulfate and ammonium sulfate, and by DEAE-Sephacel, octyl-Sepharose and puUulan-Sepharose column chromatography (12). The final enzyme solution was purified 3511-fold over the crude enzyme extract with an overall recovery of 42% and had a specific activity of 481 units/mg protein. The average molecular weight of the enzyme was 136,500 determined by gel filtration on Sephacryl S-200 and SDS-PAGE, and it had an isoelectric point at pH 5.9. It was rich in acidic and hydrophobic amino acids. The purified enzyme was quite thermostable in the absence of substrate even up to 90°C with essentially no loss of activity in 30 min. However, the enzyme lost about 40% of its original activity at 95 C tested for 30 min. The optimum tenq)erature for the action of the purified enzyme on pullulan was 90°C. However, the enzyme activity rapidly decreased on incubation at 95°C to only 38% of the maximal 30 min. The enzyme was stable at pH 3.0-5.0 and was optimally active at pH 5.5. It produced only maltotriose and no panose or isopanose from pullulan. 

The sedimentation coefficient is 3.0 S. The approximate molecular weight of the enzyme was determined by gel filtration (117) to be about... 

Quite a different form of exopolyphosphatase was purified from the vacuolar sap of S. cerevisie (Andreeva et al., 1998b). Its molecular mass determined by gel filtration was 245 kDa. This exopolyphosphatase hydrolysed PolyP3 only slightly, and its specific activity increased with the increase in PolyP chain length (Table 6.6). It was unable to hydrolyse adenosine- and guanosine-tetraphosphates and was insensitive to antibodies inhibiting the low-molecular-mass exopolyPase of the cytosol (Table 6.4). This enzyme was stimulated by divalent metal cations to a much lesser extent than 40 kDa exopolyphosphatase (Table 6.5) and was inhibited by EDTA (Table 6.4). The inhibitory effect of EDTA is explained by the binding of Co2+, which is the best activator of the vacuolar exopolyphosphatase at 0.1 mM. 

In 1972 Ogawa and Toyama (56) purified three components— A-I-a, A-I-b, and A-II-1—which were adsorbed on a gauze column during purification from Cellulase Onozuka P1500, a commercial preparation of T. viride cellulase. These three components had molecular weights of 32,000, 48,000, and 48,000 as determined by gel filtration and contained 7-16% carbohydrate. Each is reported to carry out the random hydrolysis of CM-cellulose and to degrade hydrocellulose (Avicel) and cellooligosaccharides except for cellobiose. The order of reactivity toward either cotton or Avicel was A-II-1 > A-I-b > A-I-a. The proteins adsorbed on cellulose comprised 38% of the total cellulase protein. 

Calculated from sedimentation and diffusion, unless otherwise stated. Determined by gel-filtration experiments. Assumed value. 

You have isolated and purified a new enzyme (E) which converts a single substrate (S) into a single product (P). You have determined by gel filtration as 46,400. However, in SDS gel electrophoresis, a molecular mass of 23 kDa was indicated for the single protein band observed. A solution of the enzyme was analyzed in the following way. The absorbance at 280 nm was foxmd to be 0.512. A 1.00 ml portion of the same solution was subjected to amino acid analysis and was found to contain 71.3 nmol of tryptophan. N-terminal analysis on the same volume of enzyme revealed 23.8 nmol of N-terminal alanine. 

MPO from H. niger was isolated and its molecular weight determined by gel filtration (135 kDa) and the SDS-PAGE analysis showed that the enzyme is a dimmer [140]. MPO has also been obtained from other plant speeies such as D. stramonium [141] and N. tabacum [142], Inhibition studies of Hyoscyamus MPO demonstrated the quinoprotein characteristic of this enzyme [140], a feature previously reported for N. tabacum MPO, after conducting a suicidal inhibition of the enzyme with phenyUiydrazine [143]. 

A deoxyadenosylcobalamin-dependent ribonucleoside triphosphate reductase has been partially purified from cell free extracts of the extreme thermophile, Thermus X-l 14). The enzyme preparation catalyzed the reduction of GTP and CTP at comparable rates, while UTP and ATP were reduced at only one-tenth the rate of GTP reduction. Only the dithiols could serve as reducing substrates. The enzyme has a temperature optimum of 70°, and the allosteric regulation of the enzyme activity is also temperature-dependent. The reduction of ATP is specifically stimulated by dGTP only at a higher temperature. Maximum stimulation of ATP reduction is observed at approximately 75°, while no stimulation can be detected at 37°. The molecular weight determined by gel filtration was approximately 80,000 but no information about the subunit structure is yet available. 

Tomato PG I and PG II are similar in many respects. Both enzymes are endo-PG s although PG II is more effective in reducing the viscosity of pectate (30). Their pH optima are near 4.5, and both enzymes exhibit broad peaks of activity extending from pH 1.5 to 5.5 when hydrolyzing short-chained substrates (30). They are basic glycoproteins with pH s of 8.6 and 9.4 for PG I and PG II, respectively (12). Antibodies raised against PG II react also with PG I (32). The same polypeptide is obtained when PG I and PG II are denatured in SDS solutions (12., 36). However, the enzymes differ markedly in molecular size and stability to heat. The molecular weights, as determined by gel filtrations are 100,000 and... 

The ICS (MenF) encoded by the menF gene has been overexpressed and purified to homogeneity. The purified enzyme had a relative of 48 000 " as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native as determined by gel filtration chromatography, was 98 000, thus... 

The mmB gene was cloned and its complete nucleotide sequence was determined. When the gene was overexpressed and the protein was purified to homogeneity, the subunits were found to have an of 32 000, whereas the native protein had an M,. of 112 000 as determined by gel filtration. Thus, the enzyme is a homotetramer. ... 

Figure 11. Proteolytic breakdown of casein in aging, starter-less cheese as determined by gel filtration (200).

One glycoprotein, invertase, has been studied in detail by radiation inactivation. This glycoprotein is composed of two identical 120,000 monomers each is made of a 60,000 polypeptide core, and the rest is carbohydrates (Trimble and Maley, 1977). The RIS is about 120,000 (Pollard et al., 1952 Lowe and Kempner, 1982). Removal of about 90% of the carbohydrates with endoglycosidase H reduced the Mr (determined by gel filtration) but had no effect on the RIS (Lowe and Kempner, 1982). It was concluded that a hit in the glycosylated portion of the invertase is not transferred to the polypeptide core, the sole part of the molecule apparently required for enzyme activity. 

Four isolectins have been isolated from bulbs of Crocus sativus with approximately molecular weight 48 KDa as determined by gel filtration chromatography [42]. Rivoflavine and thiamin are also constituents of saffron [43]. Very recently, anthocyanins were identified in the flowers of Crocus sativus [44],... 

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